English  Sprachen Icon  |  Gebärdensprache  |  Leichte Sprache  |  Kontakt


In a role reversal, RNAs proofread themselves

By Dr. Claus Kuhn and Dr. Jaclyn Jansen (01.04.2015)

Molecular photographs of an enzyme bound to tRNA reveal a new, inherent quality control mechanism.

Building a protein is a lot like a game of telephone: information is passed along from one messenger to another, creating the potential for errors every step of the way. There are separate, specialized enzymatic machines that proofread at each step, ensuring that the instructions encoded in our DNA are faithfully translated into proteins. The building blocks for proteins are carried by molecules known as transfer RNAs (tRNAs). tRNAs work with other cellular machinery to ensure that the building blocks – amino acids – are arranged in the proper order. But before a building block can be loaded onto a tRNA molecule, a three-part chemical sequence called “CCA” must be added to all tRNAs. These letters are added by an appropriately named machine, the CCA-adding enzyme, and they mark the tRNA as a fully functional molecule.

Intriguingly, if a tRNA is mutated, the CCA-adding enzyme duplicates its message. The letters now read “CCACCA,” signaling that the tRNA is flawed. The cell rapidly degrades the aberrant tRNA, preventing the flawed message from propagating.

But how does the CCA-adding enzyme distinguish between normal and mutant tRNAs?

Kuhn Fig. 1[Bildunterschrift / Subline]: Fig.1 A molecular photograph of the CCA-adding enzyme in complex with a mutated tRNA reveals a remarkable, new proofreading mechanism. In general, enzymatic machines are responsible for correcting errors. However, the CCA-adding enzyme (shown in purple, blue, green, and cyan) doesn’t correct errors at all. Instead, the RNA (in orange) has an in-built mechanism that allows it to proofread itself.

Using X-ray crystallography – a type of molecular photography – to observe the enzyme at work, we found that the enzyme doesn’t discriminate at all. In fact, it is the RNA that is responsible for proofreading itself.

We used bona fide tRNAs and tRNA-like noncoding RNAs to study the error-correcting mechanism. In a series of molecular photographs of the CCA-adding enzyme bound to both RNAs we found that the CCA-adding enzyme acts as a molecular vise and uses a screw-like motion before adding the CCA group to the end of the RNA. Under normal circumstances, after the addition of the final letter A, the enzyme tries to ‘twist’ the RNA molecule again, but can’t. The pressure administered by the enzyme forces the RNA to pop out of its union with the enzyme – with only a single CCA group attached.

Kuhn Fig. 2[Bildunterschrift / Subline]: Fig. 2 The CCA-adding enzyme (colors as in Fig.1) acts as a molecular vise before attaching the sequence CCA to tRNAs. After the entire CCA triplet has been added, a normal RNA dissociates from the enzyme (labeled 2a), whereas RNAs destabilized by mutations are more flexible and allow the RNA to refold while bound to the enzyme (labeled 2b). Mutated RNA is only released from the enzyme after this second cycle of CCA addition. RNAs marked by CCACCA are then rapidly degraded in the cell.

However, when a bona fide tRNA is mutated or inherently unstable (as is the case for some non-coding RNAs) the RNA structure becomes more flexible. After a single CCA addition, this flexibility allows the RNA to buckle under the increased pressure by the enzyme. The RNA extrudes a bulge and the enzyme adds an additional round of “CCA” letters. Only after this second addition cycle does the RNA dissociate.

This is a unique proofreading mechanism, since there is no difference between the two RNAs for the enzyme – it adds CCA after screw-like RNA compression regardless of what the sequence is. So it is the instability of the RNA itself that prevents future errors during protein synthesis, ensuring that proteins are made correctly.



“On-Enzyme Refolding Permits Small RNA
and tRNA Surveillance by the CCA-Adding Enzyme” by Claus-D. Kuhn, Jeremy Wilusz, Yuxuan Zheng, Peter Beal, and Leemor Joshua-Tor. Cell 160, 644-658.

Wissenschaftlicher Werdegang
  • seit 09/2014
  • Junior Research Group Leader within the Elite Network of Bavaria at the University of Bayreuth, Germany
  • 03/2010 - 07/2014
  • Postdoctoral fellow with Dr. Leemor Joshua-Tor at Cold Spring Harbor Laboratory, New York, USA
  • 10/2009 - 02/2010
  • X-ray crystallographer with Proteros Biostructures GmbH, Martinsried, Germany
  • 03/2008 - 09/2009
  • Postdoctoral fellow with Dr. Leemor Joshua-Tor at Cold Spring Harbor Laboratory, New York, USA
  • 10/2003 - 02/2008
  • PhD student with Dr. Patrick Cramer at the Gene Center of the Ludwig-Maximilians-Universität in Munich, Germany
  • 08/2002 - 07/2003
  • Master of Science in Chemistry, Department of Biochemistry and Biophysics, Stockholm University, Sweden.
  • 10/1999 - 07/2002
  • Undergraduate Studies in Biochemistry at the University of Regensburg, Germany

  • * Junior Research Group Leader within the Elite Network of Bavaria at the University of Bayreuth, Germany (2014)
  • * Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, Yale University Medical School, USA (2008-2012)
  • * Member of the Elite Network of Bavaria in the Graduate Programs ‘Protein Dynamics in Health and Disease’ and ‘Nano-Biotechnology’ (2005-2008)
  • * Publication Award from the Center of Nanoscience, Ludwig-Maximilians-Universität in Munich, Germany (2008)
  • * Römerprize from the Department for Chemistry and Biochemistry, Ludwig-Maximilians-Universität in Munich, Germany (2007)
  • * Kekulé Fellowship from the German Association of the Chemical Industry (VCI) (2004-2006)
  • * Undergraduate Fellow of the Wilhelm Narr Foundation, Kirchheim/Teck, Germany (1999-2003)

Veröffentlichungen (Auswahl)
  • * Kuhn, C.-D., Wilusz, J.E., Zheng, Y., Beal, P.A. and Joshua-Tor, L. On-enzyme refolding permits small RNA and tRNA surveillance by the CCA-adding enzyme. (2015) Cell, in press.
  • * Kuhn, C.-D. and Joshua-Tor, L. Eukaryotic Argonautes come into focus. (2013) Trends in Biochemical Sciences 38, 263-71.
  • * Wilusz, J.E., JnBaptiste, C.J., Lu, L.Y., Kuhn, C.-D., Joshua-Tor, L. and Sharp, P.A. A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. (2012) Genes and Development 26, 2392-2407.
  • * Elkayam, E., Kuhn, C.-D., Tocilj, A., Haase, A.D., Greene, E.M., Hannon, G.J., and Joshua-Tor, L. The structure of Human Argonaute-2 in complex with miR-20a. (2012) Cell 150, 100-10.
  • * Geiger, S.R., Kuhn, C.-D., Leidig, C., Renkawitz, J., and Cramer, P. Crystallization of the RNA polymerase I subcomplex A14/43 by iterative prediction, probing, and removal of multiple flexible regions. (2008) Acta Crys. Section F 64, 413-418.
  • * Kuhn, C.-D., Geiger, S.R., Baumli, S., Gartmann, M., Gerber, J., Jennebach, S., Mielke, T., Tschochner, H., Beckmann, R., and Cramer, P. Functional Architecture of RNA polymerase I. (2007) Cell 131, 1260-1272.